Analysis of Signalling Pathways by Western Blotting and Immunoprecipitation

2011 ◽  
pp. 223-232
Author(s):  
Aymen I. Idris
Keyword(s):  
Western Blotting ◽  
Signalling Pathways

scholarly journals Resistin Induces Chemokine and Matrix Metalloproteinase Production via CAP1 Receptor and Activation of P38-MAPK and NF-κB Signalling Pathways in Human Chondrocytes

2020 ◽  
Author(s):  
Cheng-Wu Zhao ◽  
Wen-Xia Song ◽  
Bo Liu ◽  
Yu-Hang Gao ◽  
Lu Ding ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
P38 Mapk ◽  
Western Blotting ◽  
Nuclear Factor Κb ◽  
Mitogen Activated Protein Kinase ◽  
Human Chondrocytes ◽  
Signalling Pathways ◽  
Enzyme Linked Immunosorbent Assay ◽  
Qrt Pcr ◽  
Mitogen Activated Protein

Abstract BackgroundResistin is an adipokine also detected higher expression in serum and synovial fluid of patients with knee osteoarthritis (KOA). Resistin is known to be related closely to insulin resistance and inflammation. However, the pathogenic role of resistin in KOA remain unclear. Purpose of the study is to investigate whether resistin induces KOA by binding to functional receptor adenylyl cyclase-associated protein 1 (CAP1) and activating the p38 mitogen-activated protein kinase (p38-MAPK) and nuclear factor-κB (NF-κB) signalling pathways in human chondrocytes. MethodsWe enrolled 103 patients with radiographic KOA and 86 healthy participants as controls. The levels of resistin in serum and synovial fluid (SF) were determined via enzyme-linked immunosorbent assay (ELISA). CAP1 expression in cartilage tissues (21 samples of KOA cartilage and 10 samples of healthy hip cartilage) was measured using immunohistochemistry (IHC), quantitative real-time polymerase chain reaction ( qRT-PCR ), and western blotting assays. Effects of resistin on chondrocytes and CAP1 were evaluated via qRT-PCR and co-immunoprecipitation . The roles of CAP1, p38-MAPK, and NF-κB signalling pathways in the development of KOA were evaluated via adenovirus-mediated CAP1 short hairpin RNA, qRT-PCR, western blotting, and ELISA. ResultsExpression of resistin in serum and SF was elevated in severe radiographic KOA. CAP1 levels were higher in KOA cartilage and were positively correlated with resistin expression. Resistin promoted increased expression of CCL3, CCL4, MMP13, and ADAMTS-4 through CAP1 receptor. Resistin also directly bound to CAP1 as confirmed by co-immunoprecipitation. CAP1 knockdown in chondrocytes attenuated resistin-induced expression of CCL3, CCL4, MMP13, and ADAMTS-4 and activation of the p38-MAPK and NF-κB signalling pathways . ConclusionsOur study shows that resistin bound to CAP1 and upregulated the expression of proinflammatory cytokines and matrix-degrading enzymes via p38-MAPK and NF-κB signalling pathways in human chondrocytes.

scholarly journals Circ_0119872 promotes uveal melanoma development by regulating the miR-622/G3BP1 axis and downstream signalling pathways

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Shuting Liu ◽  
Liang Chen ◽  
Hua Chen ◽  
Kangkang Xu ◽  
Xi Peng ◽  
...  
Keyword(s):  
Cell Lines ◽  
Uveal Melanoma ◽  
Western Blotting ◽  
Gene Set Enrichment Analysis ◽  
Luciferase Reporter ◽  
Signalling Pathways ◽  
Circular Rnas ◽  
Mtor Signalling

Abstract Background The abnormal expression of circular RNAs (circRNAs) in uveal melanoma (UM) has been revealed, but the specific underlying molecular mechanism of their association with UM development has not been fully explored. Methods The levels of circ_0119872, G3BP1 and miR-622 in UM cell lines and tissues were determined by quantitative real-time PCR (qRT-PCR) and western blotting assays. In vitro and in vivo assays were performed to investigate the function of circ_0119872 in the tumorigenesis of UM cells. The relationships among circ_0119872, miR-622 and G3BP1 were predicted using bioinformatic tools and verified by RNA-FISH, RNA pull-down and dual-luciferase reporter assays. The effects of circ_0119872 on Wnt/β-catenin and mTOR signalling pathways were determined by gene set enrichment analysis (GSEA) and western blotting. Results We found that circ_0119872 is upregulated in UM cell lines and tissues. Moreover, overexpression of circ_0119872 promotes the malignancy of UM cells, while silencing of circ_0119872 inhibits it. In addition, circ_0119872 can directly interact with miR-622 as a miRNA sponge that regulates the expression of the miR-622 target gene G3BP1 as well as downstream Wnt/β-catenin and mTOR signalling pathways. Conclusions Circ_0119872 may act as an oncogene in UM through a novel circ_0119872/miR-622/G3BP1 axis, activating the Wnt/β-catenin and mTOR signalling pathways, which in turn may provide potential biomarkers and therapeutic targets for the management of UM.

Caspases and apoptosis

2002 ◽  
Vol 38 ◽  
pp. 9-19 ◽  
Author(s):  
Guy S Salvesen
Keyword(s):  
Cysteine Proteases ◽  
Signalling Pathways ◽  
Term Survival ◽  
Mature Adult ◽  
Intracellular Signalling Pathways ◽  
Adult Cell ◽  
Caspase Family ◽  
Cell Fragments ◽  
Pro Inflammatory Cytokines

The ability of metazoan cells to undergo programmed cell death is vital to both the precise development and long-term survival of the mature adult. Cell deaths that result from engagement of this programme end in apoptosis, the ordered dismantling of the cell that results in its 'silent' demise, in which packaged cell fragments are removed by phagocytosis. This co-ordinated demise is mediated by members of a family of cysteine proteases known as caspases, whose activation follows characteristic apoptotic stimuli, and whose substrates include many proteins, the limited cleavage of which causes the characteristic morphology of apoptosis. In vertebrates, a subset of caspases has evolved to participate in the activation of pro-inflammatory cytokines, and thus members of the caspase family participate in one of two very distinct intracellular signalling pathways.

Nutrient-regulated gene expression in eukaryotes

2006 ◽  
Vol 73 ◽  
pp. 85-96 ◽  
Author(s):  
Richard J. Reece ◽  
Laila Beynon ◽  
Stacey Holden ◽  
Amanda D. Hughes ◽  
Karine Rébora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Direct Interaction ◽  
Signalling Pathways ◽  
A Cell ◽  
One Step ◽  
Higher Eukaryotes ◽  
Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting

1993 ◽  
Vol 70 (03) ◽  
pp. 438-442 ◽  
Author(s):  
B Grøn ◽  
C Filion-Myklebust ◽  
S Bjørnsen ◽  
P Haidaris ◽  
F Brosstad
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Western Blotting ◽  
Isoelectric Point ◽  
Factor Xiii ◽  
Cross Linking ◽  
2D Electrophoresis ◽  
Complex Phenomenon ◽  
Fibrinopeptide A

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.

Plasma Factor V Activation Is Prevented by Activated Protein C in the Presence of Phospholipid Vesicles, Not Platelets

1993 ◽  
Vol 69 (02) ◽  
pp. 124-129 ◽  
Author(s):  
Susan Solymoss ◽  
Kim Thi Phu Nguyen
Keyword(s):  
Western Blotting ◽  
Protein C ◽  
Thrombin Generation ◽  
Blood Platelets ◽  
Activated Protein C ◽  
Phospholipid Vesicles ◽  
Factor V ◽  
Two Stage ◽  
One Stage ◽  
Plasma Factor

SummaryActivated protein C (APC) is a vitamin K dependent anticoagulant which catalyzes the inactivation of factor Va and VIIIa, in a reaction modulated by phospholipid membrane surface, or blood platelets. APC prevents thrombin generation at a much lower concentration when added to recalcified plasma and phospholipid vesicles, than recalcified plasma and platelets. This observation was attributed to a platelet associated APC inhibitor. We have performed serial thrombin, factor V one stage and two stage assays and Western blotting of dilute recalcified plasma containing either phospholipid vesicles or platelets and APC. More thrombin was formed at a given APC concentration with platelets than phospholipid. One stage factor V values increased to higher levels with platelets and APC than phospholipid and APC. Two stage factor V values decreased substantially with platelets and 5 nM APC but remained unchanged with phospholipid and 5 nM APC. Western blotting of plasma factor V confirmed factor V activation in the presence of platelets and APC, but lack of factor V activation with phospholipid and APC. Inclusion of platelets or platelet membrane with phospholipid enhanced rather than inhibited APC catalyzed plasma factor V inactivation. Platelet activation further enhanced factor V activation and inactivation at any given APC concentration.Plasma thrombin generation in the presence of platelets and APC is related to ongoing factor V activation. No inhibition of APC inactivation of FVa occurs in the presence of platelets.

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